Hybridoma technology for the isolation of monoclonal antibodies is, in general, limited to the generation of rodent mAbs and results in the immortalisation of only a small fraction of the specific antibody-forming cells available in an immunised animal. Antibodies from bacterially expressed libraries are restricted by practical limits to the size of libraries and the requirement for the antibody to be properly folded and expressed in bacteria. In addition, antibodies generated by both these methods frequently require affinity enhancement to obtain antibodies of a high enough affinity for therapeutic use. A number of alternative methods have been designed to enable high affinity antibodies generated during in vivo immune responses to be isolated from any species (Babcook et al., 1996, Proc. Natl. Acad. Sci, 93, 7843-7848; WO 92/02551; de Wildt et al. (1997) Journal of Immunological Methods, 207:61-67 and in Catrin Simonsson Lagerkvist et al. (1995) BioTechniques 18(5):862-869.).
The first alternative method to be designed was the selected lymphocyte antibody method (SLAM) which enables a single lymphocyte that is producing an antibody with a desired specificity to be identified within a large population of lymphoid cells and the genetic information that encodes the specificity of the antibody to be rescued from that lymphocyte. Antibody producing cells which produce antibodies which bind to selected antigens are detected using an adapted haemolytic plaque assay method (Jerne and Nordin, 1963, Science, 140, 405). In this assay, erythrocytes are coated with the selected antigen and incubated with the population of antibody producing cells and a source of complement. Single antibody producing cells are identified by the formation of haemolytic plaques. Plaques of lysed erythrocytes are identified using an inverted microscope and the single antibody producing cell of interest at the centre of the plaque is removed using micromanipulation techniques. The antibody genes from the cell are cloned by reverse transcription PCR. The physical isolation of these cells limits the number of B cells which can be detected and isolated. As a result many of the antibodies isolated may still require affinity enhancement as their affinity may only be in the nanomolar range. See for example, Babcook et al., supra where an affinity of only 1.76 nanomolar (1.76×109 M−1) is described.
In the haemolytic plaque assay described above, the red blood cells are typically coated with antigen via a biotin/streptavidin coupling system that requires the antigen to be biotinylated. This method is therefore restricted to antigens that are available in a pure form and to those that can be biotinylated without affecting epitope presentation. This method clearly precludes the isolation of antibodies against certain types of antigens. For example, many proteins are difficult to purify, particularly cell surface expressed proteins, such as type III proteins. Many proteins alter their conformation and presentation of desirable epitopes upon biotinylation, for example proteins that contain lysine groups in their active site.
It may also be desirable to produce antibodies against unknown antigens, such as proteins expressed on the surface of cells, such as tumour cells and activated T cells. The direct use of tumour cells in the plaque assay instead of antigen coated erythrocytes is difficult to achieve given the requirement for cell lysis to occur in order for plaques containing antibody-producing cells to be identified. Cell lysis is dependent on cell type, antigen and antibody concentration. Red blood cells coated with the desired antigen will bind large amounts of available antibody and will lyse readily in the presence of complement. Other cell types such as tumour cells will not lyse so readily, especially when the availability of antigen on the surface may be very low and hence antibody binding will be low.
In the method of de Wildt et al., B cells from patients suffering from the autoimmune disease systemic lupus erythematosus (in which patients often produce autoantibodies against the U1A protein) were subjected to panning using culture plates coated with U1. Cells which did not bind to the U1A were removed by washing. The adhering cells were then collected from the plates using trypsin treatment and subjected to single cell sorting using a flow cytometer to select individual U1A-specific B cells. Single B cells were then cultured in 96-well plates and clonally expanded. Culture supernatants were then tested for antibody production and U1A-specific B cell clones were identified. Total RNA was then extracted from the positive wells and the VH/VL regions from the B cells were cloned.
In Catrin Simonsson Lagerkvist et al. (1995) PBMCs from tetanus-immunized patients which bound tetanus toxoid (TT) were isolated using TT-coated magnetic beads. Single, TT-specific B cells were isolated using an automatic pipette. 0.3 B cells per well were then seeded into 96-well microplates and clonally expanded. The wells were then tested for the presence of TT-specific antibodies. The V-region genes of antibodies from the positive wells were then cloned.
The methods of both de Wildt et al. and Catrin Simonsson Lagerkvist et al. require the isolation of individual B cells which recognise the antigen of interest prior to clonal expansion, which isolation may be both cumbersome and time-consuming. Also, because only one or fewer B cells are seeded in each microtitre well, a large number of microtitre plates is required and must be screened to identify those B cells which specifically recognise the antigen of interest.
Accordingly, there is a need for less labour-intensive methods for isolating antibodies with a desired function. In addition there is a need for higher affinity antibodies which do not require subsequent affinity enhancement.
The present invention provides a method for the isolation of high affinity antibodies with a desired function.